Cell immaturity and white/beige adipocyte potential of primary human adipose-derived stromal cells are restrained by culture-medium TGFβ1

Abstract

Human adipose-derived stem/stromal cells (hASCs) can differentiate into specialized cell types and thereby contribute to tissue regeneration. As such, hASCs have drawn increasing attention in cell therapy and regenerative medicine, not to mention the ease to isolate them from donors. Culture conditions are critical for expanding hASCs while maintaining optimal therapeutic capabilities. Here, we identified a role for transforming growth factor β1 (TGFβ1) in culture medium in influencing the fate of hASCs during in vitro cell expansion. Human ASCs obtained after expansion in standard culture medium (Standard-hASCs) and in endothelial cell growth medium 2 (EGM2-hASCs) were characterized by high-throughput transcriptional studies, gene set enrichment analysis and functional properties. EGM2-hASCs exhibited enhanced multipotency capabilities and an immature phenotype compared with Standard-hASCs. Moreover, the adipogenic potential of EGM2-hASCs was enhanced, including toward beige adipogenesis, compared with Standard-hASCs. In these conditions, TGFβ1 acts as a critical factor affecting the immaturity and multipotency of Standard-hASCs, as suggested by small mother of decapentaplegic homolog 3 (SMAD3) nuclear localization and phosphorylation in Standard-hASCs vs EGM2-hASCs. Finally, the typical priming of Standard-hASCs into osteoblast, chondroblast, and vascular smooth muscle cell (VSMC) lineages was counteracted by pharmacological inhibition of the TGFβ1 receptor, which allowed retention of SMAD3 into the cytoplasm and a decrease in expression of osteoblast and VSMC lineage markers. Overall, the TGFβ1 pathway appears critical in influencing the commitment of hASCs toward osteoblast, chondroblast, and VSMC lineages, thus reducing their adipogenic potential. These effects can be counteracted by using EGM2 culture medium or chemical inhibition of the TGFβ1 pathway.Abbreviations: ASCs, adipose-derived stem/stromal cells; BM-MSCs, bone marrow mesenchymal stem/stromal cells; BMP, bone morphogenetic protein; CFU-f, colony-forming unit fibroblast; EGM2, endothelial cell growth medium 2; FBS, fetal bovine serum; GO, Gene Ontology; GSEA, gene set enrichment analysis; hrTGFβ1, human recombinant transforming growth factor-beta 1; MEM, minimum essential media; MSCs, mesenchymal stem/stromal cells; PCA, principal component analysis; SMAD, small mother of decapentaplegic homolog; SVF, stromal vascular fraction;TGFβ1R, transforming growth beta 1 receptor; TGFβR1, transforming growth beta receptor type 1; UCP1, uncoupling protein 1; VSMCs, vascular smooth muscle cells.

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